TaqMan® chemistry

Background

Initially, intercalator dyes were used to measure real-time PCR products. The primary disadvantage to these dyes is that they detect accumulation of both specific and nonspecific PCR products.
Development of TaqMan® chemistry

Real-time systems for PCR were improved by the introduction of fluorogenic-labeled probes that use the 5' nuclease activity of Taq DNA polymerase. The availability of these fluorogenic probes enabled the development of a real-time method for detecting only specific amplification products.

Step-by-step process

An oligonucleotide probe is constructed containing a fluorescent reporter dye on the 5' end and a quencher dye on the 3' end. While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET).

If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5' nuclease activity of Taq DNA polymerase as this primer is extended.

This cleavage of the probe:-- Separates the reporter dye from the quencher dye, increasing the reporter dye signal.
-- Removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.

Additional reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.

SYBR® chemistry or other double-stranded DNA binding dyes

Small molecules that bind to double-stranded DNA can be divided into two classes:

Intercalators

Minor-groove binders

Regardless of the binding method, there are two requirements for a DNA binding dye for real-time detection of PCR:

Increased fluorescence when bound to double-stranded DNA

No inhibition of PCR

We have developed conditions that permit the use of the SYBR® Green I dye in PCR with little PCR inhibition and increased sensitivity of detection compared to ethidium bromide. Additionally, we have newer SYBR® Green dyes that fluoresce more brightly and inhibit PCR less than the original SYBR® Green I.

How SYBR® dye chemistry works

SYBR® dye detects polymerase chain reaction (PCR) products by binding to double-stranded DNA formed during PCR. Here’s how it works:

Step-by-step process

When SYBR® dye is added to a sample, it immediately binds to all double-stranded DNA present in the sample.

During PCR, DNA polymerase amplifies the target sequence which creates the PCR products.

SYBR® dye then binds to each new copy of double-stranded DNA.

As the PCR progresses, more PCR product is created. SYBR® dye binds to all double-stranded DNA, so the result is an increase in fluorescence intensity proportioned to the amount of PCR product produced.

Advantages of SYBR® dye

It can be used to monitor the amplification of any double-stranded DNA sequence.

No probe is required, which can reduce assay setup and running costs, assuming that your PCR primers are well designed and your reaction is well characterized.

Disadvantage of SYBR® dye

The primary disadvantage is that it may generate false positive signals; i.e., because the SYBR® dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences. Therefore, it is extremely important to have well-designed primers that do not amplify non-target sequences, and that melt curve analysis be performed.

Additional consideration

Another aspect of using DNA binding dyes is that multiple dye molecules may bind to a single amplified DNA molecule. A consequence of multiple dye binding is that the amount of signal is dependent on the mass of double-stranded DNA produced in the reaction. Thus, if the amplification efficiencies are the same, amplification of a longer product will generate more signal than a shorter one. This is in contrast to the use of a fluorogenic probe, in which a single fluorophore is released from quenching for each amplified molecule synthesized, regardless of its length.